Root-Specific and Xylem Parenchyma-Specific Promoter

ABSTRACT

The invention relates to a promoter that is provided with tissue-specific activity and is more active in xylem parenchyma cells of plant roots than it is in other cells of said plant. The inventive promoter allows transgenic plants to be produced with particular characteristics: (a) improved xylem charging and discharging processes in the root; (b) improved nitrogen supply; (c) reduced accumulation of harmful nitrogen in the root; (d) improved resistance to salt; (e) improved resistance to stress due to dry conditions; (f) improved tolerance to frost; (g) modified Na+/K+ concentration in the root; (h) greater resistance/tolerance to pathogens.

The present invention concerns a promoter and the use thereof as well astransgenic Plants.

Higher plans contain a vascular system, the xylem, in which the upwardtransport of water and mineral nutriments takes place.

The charging of the xylem in the root is regulated via the xylemparenchyma, which jackets the xylem strands. The xylem parenchyma cellsof the root thus represent an important control point for the materialuptake in the xylem. For this purpose the xylem parenchyma cells containspecific proton pumps, water channels and ion channels.

With its physiological importance for metabolism, the xylem parenchymaopens a series of possibilities for undertaking genetic changes inplants. Influence on the nutrient transport can occur for example viacertain proteins which are active primarily in xylem parenchyma.

Promotors are already known, which are active inter alia in xylemparenchyma cells. However the activity of these promoters is either notlimited exclusively to xylem parenchyma cells, or the promoters are notprimarily active in the roots. Thus the activity of theSultr2.1-promoter is also evidenced in leaf phloem (Takehashi et al.,2000), and the SOS1-promoter is also active in the terminal cells ofroot tips (Shi et al., 2002). The I2-promoter of the resistance gene I2of the tomato is active primarily in the xylem parenchyma cells of thesprout and besides this also in the root, the leaf and the tomato fruit(Mes et al., 2000). The high affility amino acid transporter AAP6 fromA. thaliana is expressed in the xylem parenchyma cells of all roots andleaf vascular system by the AAP6-promoter (Okumoto et al., 2002).RNA-Blot Analysis shows that the AAP6 gene is strongly expressed inroots, shoot and stem leaves and weakly in the sprout and in the flower(Rentsch et al., 1996).

It is thus the task of the present invention to influence metabolism ofa plant targetedly in the area of the xylem parenchyma of roots.

In accordance with the invention the set task is solved by a promoterhaving the characteristics of claim 1.

Certain terms used in this application are described in greater detailin the following:

A promoter means a nucleic acid sequence, which controls the expressionof a gene, in certain cases depending upon endogenous and exogenousfactors. These factors include for example inductors, repressors andsimilar DNA-bonding proteins, however also environmental influences. Apromoter can be comprised of multiple elements. It includes however atleast one regulator element, which is responsible for the transcriptionof a gene under its control.

A promoter which is more active in parenchyma cells of plant roots thanin other cells of the plant exhibits, for example in roots, an activitymeasurable by RNA-Blot, which is detectable in comparable experimantalconditions in above-ground organs of the plant such as petioles, leavesand flowers at less than 20%, preferably at less than 10% and inparticular at less than 5%. The specificity is not limited to aparticular experimental time, but rather is fundamentally exhibitedduring the entire vegetative time.

“Derivatives” of a promoter are shortened or elongated or segments ofidentical versions or homologs of this promoter with the same orsubstantially the same characteristics.

“Pathogen inducability” means the influence of external factors on theplant, which are followed by a defensive reaction. These could beattacks by insects (grubs), bacteria, fungus, nematodes or otherpathogens, however also abiotic influences, such as mechanical woundingor water or salt stress.

“Direct antifungal activity” means that gene products act directlyantifungally, in that they, for example, dissolve cell walls or code forphytoalexinsynthases or, as the case may be, for metabolites whichactively limit fungal metabolism.

“Indirect antifungal effect” means that gene products activate the plantgenetic defenses. These include for example resistance genes, componentsof signal transduction (such as kinases, phosphatases), transcriptionfactors or enzymes which produce signal substances (such as ethyleneforming, salicylicic acid forming or jasmonate forming enzymes, reactiveoxygen species forming enzymes, nitratemonoxide forming enzymes).

The term “infection” refers to the earliest point in time at which themetabolism of the fungus (for example the growth of the fungus) isprepared for penetration of the host tissue. This includes for examplethe outgrowth of hyphae or the formation of specific infectionstructures such as penetration hyphae and appressoria.

The expression “homology” as used herein means a homology of at least70% at the DNA-level, which can be determined by known processes, forexample computer controlled sequence comparison (S. F. Altschul et al.,(1990), Basic Local Alignment Search Tool, J. Mol. Biol. 215: 403-410).

“Complimentary nucleotide sequence” means, with reference to a doublestranded DNA, that the second DNA strand exhibits the nucleotide basescomplimentary—according to the base pairing rules—to the first DNAstrand, which correspond to the bases of the first strand.

The term “hybridized” used herein means hybridizing under conventionalconditions, as described in Sambrook et al. (1989), preferably understringent conditions. Stringent hybridization conditions are forexample: hybridizing in 4×SSC at 65° C. and subsequent multiple washingin 0.1×SSC at 65° C. for a total of approximately one hour. Lessstringent hybridization conditions are for example: hybridizing in 4×SSCat 37° C. and subsequent multiple washing in 1×SSC at room temperature.“Stringent hybridization conditions” can also mean: hybridizing at 68°C. in 0.25 M sodiumphosphate, pH 7.2, 7% SDS, 1 mM EDTA and 1% BSA for16 hours and subsequent two times washing with 2×SSC and 0.1% SDS at 68°C.

The invention is described in greater detail in the following withreference to the figures and examples.

The inventive promoter is active in xylem parenchyma cells of plantroots. No activity or only a small amount of activity is detectable inthe above ground organs of the concerned plant. This characteristic canbe used for improving the charge and discharge processes of the xylem ofthe root and therewith can be used for improving the metabolism. Byusing the promoter it is thus also possible to provide transgenic plantsand parts of these plants such as seeds.

The inventive promoter can be used to improve the nitrogen exchange ofthe plants. For this, transport protein genes for nitrate and aminoacids are overexpressed in the root xylem parenchyma cells and theloading of the xylem is fortified with the N-compounds. A furtherimprovement in the N-metabolism is provided by the reduced storage of“harmful nitrogens” in the storage organs of the plant. Elevatedconcentrations of nitrogen compounds in storage organs often reduce thenutrient physiological value of harvested products, or impede theisolation of stored compounds such as sucrose from sugar beet roots. Thereduced storage of “harmful nitrogen” in the root can also be achievedby an amplified deposit of amino acids in the xylem and transporting inthe above-ground plant organs.

The inventive promoter can be used to improve water stress tolerance ofthe plants. The concentration of the phytohormone abscisic acid (ABA)increases in the roots in reaction to the drying of the soil. ABA istransported from the roots via xylem into the leaves, where it causes aclosing of the stomata. By a controlling the expression of theABA-transporter in the xylem parenchyma cells the ABA entry into xylemcan be regulated and the drought stress tolerance of the plants can beimproved.

The inventive promoter can be used to reduce the concentration of thecations sodium (Na+) and potassium (K+) in the roots of sugar beets. TheNa+/K+ concentration determines the processing quality of sugar beetswith respect to the technical extraction of sugar (Schlweck et al.,1984). The Na+ and K+ transport molecules in the xylem parenchyma cellsof the root have a key job for a low Na+/K+ concentration in the sugarbeet root. With the aid of the promoter suitable processes can becarried out, in order to keep the Na+/K+ concentration low, for examplethe overexpression of the Na+/H+ Antiporter SOS1 (Shi et al., 2002) inthe xylem parenchyma cells. On the other hand, the loading of the xylemK+ is regulated separately from the K+ uptake from the soil. While AKT1is responsible for the K+ uptake from the medium, the K+ loading of thexylem occurs via the transport molecule SKOR (stelar K+ outwardrectifier). The targeted overexpression of SKOR (Gaymard et al. 1998)would lead to an amplified transport of K+ out of the roots.

The inventive promoter can also be used to improve the diseaseresistance of plants. Numerous soil inhabiting fungus of this speciesFusarium oxysporum or Verticillium spp. use the xylem for spreading inthe plant. By combination of the pathogen inducible promoter with agene, of which the gene product has a direct or indirect antifungaleffect, the further spreading of the fungus in xylem can be prevented,and accordingly be fungus resistance can be realized. Herein thepathogen inducibility of the promoter takes on a particular role, inorder to achieve in the xylem parenchyma a level of expression criticalfor the effectiveness of the antifungal working principle.

Viral infections of the sugar beet are often limited to one organ suchas the root or the above-ground plant parts. Thus the virus BMYVVinfects and colonizes predominantly the sugar beet root, and theyellowing virus BMYV and BYV are only found in the leaves. The rootspecific promoter can thus be used to organ-specifically translate orconvert the virus resistance concepts involving gene silencing or as thecase may be the antisense technique.

FIG. 1 shows the root specific expression of the gene 2-1-88 by aRNA-Blot experiment. Respectively 10 μg whole cell-RNA, which wereisolated from the organs of 2-year old flowering sugar beet, wereseparated in a denaturing formaldehyde agarosegel. RNA was isolated fromthe leaves, the plant root, the sprout and the flowers. The smart cDNAfragment 2-1-88 was used as hybridization testing probe.

FIG. 2 shows a reporter gene vector 2-1-88-GUS with a translationalfusion between the Promoter (SEQ ID NO: 1) and the GUS-gene from E.coli. The promoter in the vector 2-1-88-GUS was isolated as HindIII-NcoIfragment and includes the nucleotide positions 1-2502 of the nucleotidesequence SEQ ID NO: 1.

FIG. 3 shows a reporter gene vector 2-1-88-LUC with a translationalfusion between the promoter (SEQ ID NO: 1) and the luciferase gene fromPhotinus pyralis. The promoter 2-1-88 in the vector 2-1-88-LUC includesthe nucleotide positions 1-2502 of the nucleotide sequence SEQ ID NO: 1.

FIG. 4 shows a modular organization of the promoter (SEQ ID NO: 1) andthe distribution of cis-elements.

FIG. 5 shows a binary vector 2-1-88-luc-kan, which was used for sugarbeet transformation.

FIG. 6 shows a specific luciferase activity of the reporter geneconstruct 2-1-88-luc-kan in extract from roots and leaves of younger andolder transgenic sugar beets (WP4) as well as the activity of thenontransgenic starting line (control). The scaling of Y-axis islogrithmic.

FIG. 7 shows a spatial distribution of the luciferase activity in theroot cross section of the transgenic sugar beet line WP4-15 incomparison to nontransgenic starting line (control). The root discs werephotographed under illumination (left) and then the light emission fromthe root disc in the dark was documented (right). The position andstrength of the light emission correlated with the activity of thePromoter (SEQ ID NO: 1) in the root of the transformant WP4-15 (rightbottom). The nontransgenic starting line shows no light emission (rightupper).

FIG. 8 shows a microscopic detail image of the transgenic root discWP4-15 from FIG. 7. In the left image half the illuminated root surfacecan be seen. The vascular bundle comprised of phloem and xylem appearsas dark slits. In the right image half the localization of the lightemission about the xylem bundle is demonstrated. This image was recordedin the dark, so that only the light emission from the object wasmeasured.

FIG. 9 shows a super-positioning of the left and the right image halvesfrom FIG. 8. The luciferase reporter gene activity appears red in thisfigure. The reporter gene activity is limited to the cell layer betweenxylem and phloem or as the case may be the immediate vicinity about thexylem and the xylem parenchyma cells.

FIG. 10 shows a reporter gene vector pUB1-minimal with the minimalpromoter of the ubiquitine promoter from corn.

CHARACTERIZING THE PROMOTER 2-1-88

The promotor (SEQ ID NO: 1) originated from a—previously unknown—sugarbeet gene 2-1-88 and is in the following also referred to as 2-1-88promoter. The analysis of this promoter with the aid of the PLACE databank (Higo et al., 1999) shows that numerous cis-elements for pathogeninduceability, root specificity as well as water and cold stressresponsivity can be demonstrated (Table 1). One characteristic pathogenresponsive element is W-Box, which occurs once as “classical” W-Box(Rushton et al., 2002) and four times as W-Box NPR1 (Yu et al., 2001) inthe promoter (FIG. 4). Besides the core motif pattern TTGACC or as thecase may be TTGAC, respectively the 15 nucleotides upstream anddownstream of the W-core motif have important significance for thepathogen inducibility in combination with the core motif. Beyond this,the Box-A from the pathogen-inducible PcPAL promoter is present. Theroot specificity of the promoter is determined by the numerous rootspecific motives (root Box ATATT) and the cis-elements forauxinresponsivity (Aux-IAA4 and Aux-SAU15A) (Guilfoyle et al., 1998).The water, salt and cold stress inducibility of the promoter can betraced back to the presence of the MYC and MYB bonding sites (Abe etal., 2003), which are involved in the ABA signal transduction.

Fusion of the 2-1-88-Promoter with the Luciferice Gene of PhotinusPyralis

In order to demonstrate the activity of the 2-1-88 promoter in sugarbeets, the promoter was translationally fused in the luciferase genefrom photinus pyralis and transformed in sugar beets. For this thevictor 2-1-88-GUS was first linearized by a SacI cleavage withsubsequent T4-DNA-Polymerase-replenishing reaction. By subsequentdigestion with NcoI the GUS-gene was released. In the thus-preparedvector the luciferase gene from photinus pyralis (Promega, MannheimGermany) was cloned as NcoI-BgIII (replenished) fragment. The resultingvector carried the characteristic 2-1-88-LUC (FIG. 3) and contained atranslational fusion between the 2-1-88 promoter and the luciferasegene.

For the transformation of the sugar beet the expression cassettecomprised of the 2-1-88-promoter and the luciferase gene was cloned overin the binary vector pGPV-kan (Becker et al., 1992). For this the2-1-88-promoter-luciferase gene-combination with PvuII and HindIII wascut out of the vector 2-1-88-LUC and cloned in the binary vectorpGPTV-kan linearized with HindIII and SmaI. The resulting vector wasgiven designation 2-1-88-luc-kan (FIG. 5). The vector 2-1-88-luc-kan wastransformed in the agrobacterium tumefaciens line C58C1 with theresident plasmid pGV2260 by a direct DNA-transformation process (An,1987). The selection of recombinant A. tumefaciens clones occurred byuse of the antibiotic kanamycin (50 mg/l).

The transformation of the sugar beets occurred according to Lindsey etal. (1991) with use of the antibiotic kanamycin. The transgenesisity ofthe plants was verified by PCR. The use of the primaryGTGGAGAGGCTATTCGGTA and CCACCATGATATTCGGCAAG lead to the amplificationof a 553 bp DNA fragment from the nptII gene. The PCR was carried outusing 10 ng genomic DNA, a primer concentration of 0.2 μM at anannealing temperature of 55° C. in a Multicycler PTC-200 (MJ Research,Watertown, USA).

Confirmation of the 2-1-88 Promoter Activity in Roots of TransgenicSugar Beets

Transgenic sugar beets, which were transformed with the reporter genecontruct 2-1-88-luc-kan, were raised in greenhouse conditions. Theactivity of the promoter was analyzed in roots and leaves of young andold sugar beets by reporter gene measurements.

The Photinus pyralis-Luciferase activity was determined using aLuciferase Assay System (Promega, Mannheim, Germany) in a SiriusLuminometer (Berthold Detection System GmbH, Pforzheim, Germany) inaccordance with the manufacturer's specifications. For extracting anenzyme extract suitable for the measurements, first the weight of thetissue sample was determined. The leaf samples were homogenized withaddition of sea sand with a 10-fold volume (v/w) of Passive Lysis Buffer(PLB) in a mortar and the root sample was homogenized in a conventionalkitchen apparatus (Warring Blender). The liquid supernatant wastransferred into a 1.5 ml-Eppendorf vessel and centrifuged 5 min at 4°C. and 20,000 g. The clear supernatant was removed and respectively 10μl raw extract were introduced for the Photinus luciferase activitymeasurement.

While the promoter (SEQ IDS NO: 1) was strongly, or as the case may bevery strongly, expressed in the young and the old roots of 9 independenttransformants, and the reporter gene activity in 7 transformants (WP4-2,WP4-5, WP4-6, WP4-8, WP4-9, WP4-12, WP4-16), in comparison to thenontransgenic starting line in which it was hardly demonstratible inyoung plants and not in old plants (FIG. 6). The promoter (SEQ ID NO: 1)was primarily expressed in the root of the sugar beet according to theresults of the reporter gene study which correlated with theRNA-blot-study.

The 2-1-88 Promoter is Speficially Active in the Xylem Parenchyma Cellsof the Sugar Beet Root

In order to analyze the spatial distribution of the promoter activity inthe sugar beet roots, transverse and longitudinal sections of the beetwere prepared and the root sections incubated 2-4 hours in a solution of100 μM luciferin plus 5% DMSO at room temperature. Subsequently thelight emission from the root sections, which can be traced back to theluficerase activity, were detected with the aid of a MicroMAX DigitalCCD Camera (Visitron Systems GmbH, Puchheim, Germany).

The analysis of the cross sections showed that the promoter activity isassociated with the position of the vascular cells in the individualcambriun rings of the root (FIG. 7). For a detailed analysis thestereo-microscope Stemi 2000 (Zeiss, Germany) was equipped with a camerawith the aid of HRD microscope adaptor. According to the microscopicanalysis the promoter activity is limited to the xylem parenchyma cellsbetween xylem and phloem or as the case may be the xylem parenchymacells surrounding the xylem (FIG. 8 and 9).

With the inventive promoter, transgenic plants with particularcharacteristics can be produced:

a. improved charging and discharging processes of the xylem in the root,

b. improved nitrogen uptake,

c. reduced accumulation of “harmful nitrogen” in the root,

d. improved salt resistance,

e. improved drought stress resistance,

f. improved frost tolerance,

g. improved Na+/K+ concentration in the root, and

h. elevated resistance/tolerance to pathogens.

The 2-1-88 Promoter is Induced in the Xylem Parenchyma Cells by Bioticand Abiotic Stress

Transgenic WP4 sugar beets, which were transformed with the reportergene construct 2-1-88-luc-kan, were infected with the parasitic fungusFusarium oxysporum f. sp. betae. Fusarium oxysporum f. sp. betae is avascular pathogen of beet, which propagates in the xylem system of theplant and causes fusarium deadhead. Four weeks after infection, theluciferase reporter gene activity in the infected root was quantified.The reporter gene activity was significantly increased in the infectedroots in comparison to the noninfected transgenic plants. The2-1-88-promoter was less induced by pathogen infestation. The analysisof the special distribution of the reporter gene activity with the aidof the CCD-camera showed that the promoter activity was significantlyhigher in the xylem parenchyma cells which surrounded the infected xylembundle.

Change of the Nitrogen Metabolism of Plants

The nitrogen metabolism of plants can be improved in many respects bythe use of the inventive promoter. The specific elevation or reductionof the number of suitable transport proteins in the xylem parenchymacells of the root improved the uptake and the transport of N-compoundsin the plant.

By the root-specific expression of transport protein genes for nitrateions in xylem parenchyma, the nitrogen uptake from the soil can beincreased and the utillization of N-fertilizers can be improved. Theimproved nitrate transport in the above-ground plant parts leads to anelevated amino acid production in the leaves. A more efficientutilization of the N-compounds already reduced to amino acids in theroots serves, or is served by, the xylem parenchyma specific expressionof amino acid transporters by the promoter.

A further improvement of the N-metabolism results from the reducedstorage of “harmful nitrogen” in the storage organs of the plant.Elevated concentrations of N-compounds in the storage organs oftenreduce the nutrient physiological value of harvested products orcomplicate the extraction of stored substances such as sucrose fromsugar beet roots. A reduced storage of “harmful nitrogen” in the sugarbeet root can be accomplished by the amplified transport of amino acidsout of the root into the above ground plant parts via the xylem. Forthis, appropriate amino acid transporters are overexpressed.

Increasing the Tolerance to Phytopathogenic Viruses

Numerous phytopathogenic virus of sugar beet have an organ specificity,that is, the viral replication occurs generally not in the entire plant,but rather only in a particular organ or tissue-type. Likewise thedamage, brought about by the viral infection, as a rule is limited tothe afflicted organ. Viral disease inducers of sugar beet with organspecificity include for example BNYVV with the preference for roots andBNYV and BYV with the limitation to beet leaves.

The inventive promoter can be used to develop a root specific BNYVVresistance in sugar beet. For this, for the transformation of thegene-silencing-dependent virus resistance strategy, a native or mutatedDNA partial sequence of the viral BNYVV-genome was combined with thepromoter. The combination of the promoter sequence and the viral DNAsequence was so designed, that the transcription of the BNYVV sequencelead to a gene silencing effective against the BNYVV. The effectivenessof the adoption of the approach was determined via a determination ofthe virus titer in the plants with utilization of an ELISA-test, whichis directed against the coat protein of BNYVV.

Increased Tolerance of Transgenic Plants Against Soil PropagatingPhytopathogenic Fungi

The inventive promoter can also be used, in combination with a gene or agene combination, to impart a direct or indirect antifungal effect inthe roots of plants. The antifungal effect leads to an elevated fungalresistance or fungal tolerance particularly against vascular parasitessuch as Fusarium oxysporum spp.

For this the promoter is fused with genes of the pathogen defense, ofwhich the gene product have a direct or indirect antifungal effect,translationally or transcriptionally. The promoter gene combinations arecloned in a binary transformation vector pGPTV and transformed by A.tumefaciens mediated transformation in sugar beet, potato or rape. Thetransgenicity of the plants was verified by PCR as already described andthe expression of the gene and the roots was verified by RNA-blotstudies. The elevated fungal resistance of the plants was observed infungal resistance tests.

Suprisingly, the root- and xylem parenchyma-specific induced expressionof pathogen defense did not lead to dwarfism or reduced tolerance oftenobserved in the case of constitutive expression in the total plant (Heiland Baldwin, 2002). A further advantage of the essentially root specificgene expression is that the resistance imparting gene product is formedonly in the organ to be protected. Commercially important plant partssuch as rape seed thus remain free of any transgenic products.

Genes, of which the products have an indirect, antifungal effect,require a particularly careful expression control, in order to avoidnegative consequences of an undesired activation of the plant defensemechanism. One example for an indirect, antifungal effect is the effectwhich stems from the coexpression of the plant resistance-R-gene incombination with an avirulence-(avr)-gene or the overexpression of anauto-activated R-gene in a plant cell. The join expression of R andavr-genes or the singular expression of an auto-activated R-gene leadsto an intensive activation of the plant defense mechanism and requires astrict regulation. The regulation is achieved, in that either the R-geneor the avr-gene is placed under the control of the inventive promoter.

Use of the Inventive Promoter in Monokotyledons

Although the promoter is also active in monokotyledons, an optimalactivity of the promoter in monokotyledons is achieved when a sugar beetminimal promoter inclusive TATA-Box is replaced by a minimal promoter ofthe ubiquitin promoter from corn inclusive the intron (SEQ ID NO: 2).For this, the SEQ ID NO: 1 from Position 1-2459 is amplified by PCR andcloned with the vector pUBI-minimal cleaved with SmaI (FIG. 10).

REFERENCES

-   Abe, H., Urao, T., Ito T. Seki, M., Shinozaki, K., and    Yamaguchi-Shinozaki K. (2003). Arabidopsis AtMYC2 (bHLH) and AtMYB2    (MYB) function as transcriptional activators in abscisic acid    signaling. Plant Cell. 15(1) :63-78.-   Altschul, S. F. et al. (1990). Basic Local Alignment Search Tool, J.    Mol. Biol. 215: 403-410-   An, G. (1987). Binary Ti vectors for plant transformation and    promoter analysis. Methods Enzymol.153, 292-305.-   Becker D, Kemper E, Schell J. and Masterson R. (1992). New plant    binary vectors with selectable markers located proximal to the left    T-DNA border. Plant Mol Biol. 20(6): 1195 7.-   Elmayan, T. and Tepfer M. (1995). Evaluation in tobacco of the organ    specificity and strength of the roID promoter, domain A of the 35S    promoter and the 35S2 promoter. Transgenic Res. 4(6):388-96.-   Gaymard F., Pilot G., Lacombe B., Bouchez D, Bruneau D, Boucherez    J., Michaux-Ferriere N., Thibaud J B, Sentenac H. (1998).    Identification and disruption of a plant shaker-like outward channel    involved in K+ release into the xylem sap. Cell 94, 647-655.-   Guilfoyle, T., Hagen, G., Ulmasov, T., and Murfett, J. (1998). How    does auxin turn on genes? Plant Physiol.118(2):341-7.-   Heil, M. and Baldwin, I. T. (2002). Fitness costs of induced    resistance: emerging experimental support for a slippery concept.    Trends Plant Sci. 2002 Feb;7(2):61-7.-   Higo, K., Ugawa, Y., Iwamoto, M. and Korenaga, T. (1999). Plant cis    acting regulatory DNA elements (PLACE) database:1999. Nucleic Acid    Research 27: 297-300.-   Lindsey, K,. Gallois, P., and Eady, C. (1991) Regeneration and    transformation of sugar beet by Agrobacterium tumefaciens. Plant    Tissue Culture Manual B7: 1-13; Kluwer Academic Publishers.-   Logemann, E., Parniske. M., Hahlbrock, K. (1995). Modes of    expression and common structural features of the complete    phenylalanine ammonia-lyase gene family in parsley. Proc Natl Aced    Sci U S A. 1995 92(13):5905-9.-   Mes J J, van Doom A A, Wijbrandi J. Simons G., Cornelissen B J, and    Haring M A. (2000). Expression of the Fusarium resistance gene 1-2    colocalizes with the site of fungal containment. Plant J. 23(2):    183-93.-   Okumoto S., Schmidt R., Tegeder M, Fischer W N, Rentsch D, Frommer W    B, and Koch W. (2002). High affinity amino acid transporters    specifically expressed in xylem parenchyma and developing seeds of    Arabidopsis. J Biol Chem. 277(47):45338-46.-   Rentsch D., Hirner, B., Schmelzer, E., and Frommer W. B. (1996).    Salt stress-induced proline transporters and salt stress-repressed    broad specificity amino acid permeases identified by suppression of    a yeast amino acid permease-targeting mutant. Plant Cell.    1996.Aug;8(8):1437-46.-   Rushton, P. J., Reinstadler. A., Lipka. V, Lippok, B., and Somssich,    I, E. (2002). Synthetic plant promoters containing defined    regulatory elements provide novel insights into pathogen- and    wound-induced signaling. Plant Cell.14(4):749-62.-   Sambrook, J., Fritsch, E. F., and Maniatis, T (1989). In Molecular    Cloning, A Laboratory Manual (Cold Spring Harbor Laboratory Press,    New York).-   Schiweck, H., Kozianowski, G., Anderlei, J. and Burba, M. (1984).    Errechnung der Dicksaft-Nichtzuckermasse aus Rübenanalysen.    Zuckerindustrie 119, 268-282.-   Shi H. Quintero F J, Pardo J M, Zhu J K. (2002). The putative plasma    membrane Na(+)/H(+) antiporter SOS1 controls long-distance Na(+)    transport in plants. Plant Cell 14, 465-477.-   Takahashi H. Watanabe-Takahashi A, Smith F W, Blake-Kalff M,    Hawkesford M J, and Saito K. (2000). The roles of three functional    sulphate transporters involved in uptake and translocation of    sulphate in Arabidopsis thaliana. Plant J.23(2):171-82.

Yu, D., Chen, C., and Chen, Z. (2001). Evidence for an important role ofWRKY DNA binding proteins in the regulation of NPR1 gene expression.Plant Cell. 2001 (7):1527-40. TABLE 1 Organization of the cis-Elementsin the Promoter 2-1-88 Cis-Element (Sequence) Position¹ Function TATABox 36-41 (+) Binding site of the basal transcriptionfactor (TATAAA)complex Root-Box 84-88 (−), 287-291 (+) Element for root specificityfrom the rol D (ATATT) 295-299 (+), 296-300 (−) promoter 341-345 (−),415-419 (−) (Elmayan and Tepfer, 1995). 455.458 (−), 587-591 (−) 627-631(−), 678-682 (+) 789-794 (−), 986-990 (+) MYC 166-171 (−) MYBCore-bonding site from the drought stress Consensus 794-799 (−)responsive promoter rd22 and other genes (CANNTG, 825-830 (−) (CBF3),involved in the ABA- and cold signal N = A, T, C, G) transduction (Abeet al., 2003) W-Box 222-227 (+) Core-motif of WRKY bonding site ofPcPR1- (TTGACC) 1 and PcPR1-2, sufficient for the pathogen induction ofsynthetic promotors (Rhushton et al., 2002) PAL-BoxA 364-369 (−) Box Afrom the pathogen responsive PcPAL (CCGTCC) 574-579 (+) promoter(Logemann et al., 1998) Aux-IAA4 490-497 (−) Auxin responsivecis-element from PS (G/TGTCCCAT) IAA4/5 promoter per Gullfoyle et al.(1998) W-Box NPR1 804-808 (+) Core-motive of the WRKY bonding site of(TTGAC) 1023-1027 (−) AtNPR1, responsible for pathogen induction2269-2273 (−) (Yu et al., 2001) 2448-2452 (+) Aux-SAU15A 825-830 (+)Auxin responsive cis-Element from (CATATG) SAUR15A Promoter perGuilfoyle et al. (1998) MYB1AT 1100-1116 (+) MYB bonding site from thedrought stress (A/TAACCA) 1170-1175 (−) responsive promoter rd22,involved in the ABA- signal transduction (Abe et al., 2003).The indicated positions are with reference to the transcription startingpoint (+1).(+) = direct Orientaton to the TATA-Box,(−) = reverse Orientation to the TATA Box.

1. A promoter which exhibits a tissue specific activity and is moreactive in xylem parenchyma cells of plant roots than in other cells ofthe plant.
 2. The promoter according to claim 1, wherein the activity ofthe promoter in xylem parenchyma cells of plant roots exhibits anactivity measureable by RNA-blot, which is detectable at less than 20%,under the same experimental conditions in above-ground organs of theplant such as petioles, leaves and flowers.
 3. A promoter, including anucleotide sequence according to SEQ ID NO: 1 or a nucleotide sequencecomplementary to the nucleotide sequence according to SEQ ID NO: 1 or anucleotide sequence, which hybridizes with the nucleotide sequenceaccording to SEQ ID NO: 1 or a nucleotide sequence complementary to thenucleotide sequence according to SEQ ID NO:
 1. 4. A derivative of apromoter according to claim
 3. 5. A vector or mobile genetic element,containing a promoter or derivative according to claim
 1. 6. Eukaryoticor prokaryotic root cells, containing a promoter or derivative accordingto claim
 1. 7. A transgenic plant or part thereof, containing a promoteror derivative according to claim
 1. 8. Transgenic plant according toclaim 7, wherein the plant is Beta vulgaris.
 9. Seeds of plantsaccording to claim
 7. 10. A method for producing a transgenic plant withone or more of the following characteristics: a. improvedloading/charging and unloading/discharging processes of the xylem in theroot, b. improved nitrogen supply, c. reduced accumulation of“detrimental nitrogen” in the root, d. improved salt resistance, e.improved drought resistance, f. improved frost tolerance, g. modifiedNa+/K+ concentration in the root, h. elevated resistance/tolerance topathogens, said method comprising transforming a plant with a promoterwhich exhibits a tissue specific activity and is more active in xylemparenchyma cells of plant roots than in other cells of the plant. 11.The promoter according to claim 2, wherein the activity of the promoterin xylem parenchyma cells of plant roots exhibits an activitymeasureable by RNA-blot, which is detectable at less than 10% under thesame experimental conditions in above-ground organs of the plant such aspetioles, leaves and flowers.
 12. The promoter according to claim 2,wherein the activity of the promoter in xylem parenchyma cells of plantroots exhibits an activity measureable by RNA-blot, which is detectableat less than 5% under the same experimental conditions in above-groundorgans of the plant such as petioles, leaves and flowers.
 13. Seeds ofplants according to claim
 8. 14. A vector or mobile genetic element,containing a promoter or derivative according to claim
 2. 15. A vectoror mobile genetic element, containing a promoter or derivative accordingto claim
 3. 16. A vector or mobile genetic element, containing apromoter or derivative according to claim
 4. 17. A transgenic plant orpart thereof, containing a promoter or derivative according to claim 2.18. A transgenic plant or part thereof, containing a promoter orderivative according to claim
 3. 19. A transgenic plant or part thereof,containing a promoter or derivative according to claim
 4. 20. Eukaryoticor prokaryotic root cells, containing a promoter or derivative accordingto claim 2.